The present invention relates to methods and apparatus for processing samples in biological sample containers such as well plates, such as imaging of the samples.
Biological samples such as animal cells are commonly cultured in containers such as well plates, omni trays, Q-trays and Petri dishes. Much of the processing of the samples can be performed automatically using robotic apparatus that can deliver containers to and from various stations at which the samples can be observed and imaged using camera equipment, and transferred to other containers using an array of pins on a movable mechanical head.
For successful imaging, it is necessary to be able to accurately position the sample in the field of view of an imaging camera, and to focus the camera on the cells of interest. Also, for general imaging, the cells need to be evenly illuminated for a good quality image, whereas in the case of observations such as fluorescence imaging, one needs to be able to focus the beam of light used to excite the fluorescence onto the relevant cells. For focusing applications, autofocus systems are preferable owing to the automated nature of the robotic apparatus.
U.S. Pat. No. 6,130,745 [1] and U.S. Pat. No. 6,441,894 [2] describe a technique for focussing a beam of laser light used to excite fluorescence in cells cultured in wells in a well plate. It is important to accurately position a tightly focussed beam within the cell colony so as to avoid exciting fluorescence in unbound fluorescent markers outside the colony. The method involves focussing the laser beam near the lower surface of the base of a well, and detecting light reflected back. The focal point is scanned upwards along the vertical axis of the well. The reflected light reaches a maximum when the light is focussed on the surface because scattering of the light is reduced at this point. Thus, the lower surface of the well base is detected. The thickness of the base (as given by the well plate manufacturer) is then added to this position so that the focussed spot can be moved to a point just inside the well, above the base, where the cell colony is located. Accuracy of the technique depends on the quality of the well plate; unknown variations in the base thickness from well to well will affect how the spot is positioned with respect to the upper surface of the base in each well. An alternative arrangement to avoid this issue which involves moving the focussed spot down onto the upper surface of the well base is more difficult to implement. The reflected signal is weaker owing to the refractive index change at the surface boundary being reduced by the fluid in the well.
With regard to focusing a camera to image the cells, a standard autofocus system may be adequate. However, for a container requiring many images, such as a well plate comprising 96, 384 or 1536 wells, it is very time-consuming to refocus the camera for each well. This is particularly problematic if no stains or fluorescent tags are used to highlight the cells; the visual contrast between the cells and their surroundings can be insufficient for the optical feedback in the autofocus system to function efficiently. As an example, under these conditions it can take over an hour to image each well in a 96-well plate by refocusing the camera for every well. A laser range finder system could be used to locate the required focal point for each well, but this is also relatively slow, and very costly.
Other problems associated with imaging samples in containers such as well plates include difficulties in illuminating the samples for non-fluorescence imaging, where any shadows cast across the samples are undesirable, and the time and complexity involved in imaging a container containing many samples (such as a well plate with many wells) where each sample must be accurately aligned with the imaging equipment.